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Auxenochlorella protothecoides is a known thiamine auxotroph but our past work has shown that it can synthesize thiamine if provided with the precursor molecule 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). Partial thiamine auxotrophy is common in microalgae with important ramifications for global phytoplankton productivity as well as engineering applications of algae. While thiamine deficiency can greatly depress algae growth and lipid content, the detailed metabolic impacts of thiamine deficiency are not well understood. We used metabolomics to study the response to thiamine-limited and replete conditions in mixotrophic A. protothecoides. We also investigated the impacts of exogenous HMP addition and the use of spent medium from another green algae, C. sorokiniana, as a source of thiamine metabolites. This is the first study, to our knowledge, that addresses metabolic impacts of thiamine deficiency and alleviation in green microalgae. Thiamine deficient cultures exhibited accumulation of pyruvate and α-ketoglutarate, indicating bottlenecks at the pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) complexes. Both PDH and OGDH require thiamine pyrophosphate (TPP) as a cofactor. Transketolase also requires TPP but we only observed build-up of ribose-5-phosphate when glucose was supplied as a substrate. As expected, thiamine and HMP addition could alleviate these metabolic bottlenecks while greatly increasing algal growth, neutral lipid and starch content. Spent medium from C. sorokiniana only appeared to partially alleviate thiamine deficiency and resulted in build-up of isocitrate and glycolate, metabolites that appeared relatively unaffected by the presence or absence of thiamine. Interestingly, longer culture time of C. sorokiniana when preparing the spent medium led to much higher availability of thiamine metabolites. Thus, under the right conditions, it may be possible to co-culture mutually beneficial algae species and/or recycle spent cultivation medium to overcome auxotrophy in algae.

Lipid productivity and fatty acid composition are important metrics for the production of high quality biodiesel from algae. Our previous results showed that co-culturing the green alga Chlorella minutissima with Escherichia coli under high-substrate mixotrophic conditions enhanced both culture growth and crude lipid content. To investigate further, we analyzed neutral lipid content and fatty acid content and composition of axenic cultures and co-cultures produced under autotrophic and mixotrophic conditions. We found that co-culturing C. minutissima with E. coli under high substrate conditions (10 g/L) increased neutral lipid content 1.9- to 3.1-fold and fatty acid content 1.5- to 2.6-fold compared to equivalent axenic C. minutissima cultures. These same co-cultures also exhibited a significant fatty acid shift away from trienoic and toward monoenoic fatty acids thereby improving the quality of the synthesized fatty acids for biodiesel production. Further investigation suggested that E. coli facilitates substrate uptake by the algae and that the resulting growth enhancement induces a nitrogen-limited condition. Enhanced carbon uptake coupled with nitrogen limitation is the likely cause of the observed neutral lipid accumulation and fatty acid profile changes.

Lipid quantitation is widespread in the algae literature, but popular methods such as gravimetry, gas chromatography and mass spectrometry (GC-MS), and Nile red cell staining suffer drawbacks, including poor quantitation of neutral lipids, expensive equipment, and variable results among algae species, respectively. A high-throughput microplate assay was developed that uses Nile red dye to quantify neutral lipids that have been extracted from algae cells. Because the algal extracts contained pigments that quenched Nile red fluorescence, a mild bleach solution was used to destroy pigments, resulting in a nearly linear response for lipid quantities in the range of 0.75 to 40 μg. Corn oil was used as a standard for quantitation, although other vegetable oils displayed a similar response. The assay was tested on lipids extracted from three species of Chlorella and resulted in close agreement with triacylglycerol (TAG) levels determined by thin layer chromatography. The assay was found to more accurately measure algal lipids conducive to biodiesel production and nutrition applications than the widely used gravimetric assay. Assay response was also consistent among different species, in contrast to Nile red cell staining procedures.

Chlorella minutissima was co-cultured with Escherichia coli in airlift reactors under mixotrophic conditions (glucose, glycerol, and acetate substrates) to determine possible effects of bacterial contamination on algal biofuel production. It was hypothesized that E. coli would compete with C. minutissima for nutrients, displacing algal biomass. However, C. minutissima grew more rapidly and to higher densities in the presence of E. coli, suggesting a symbiotic relationship between the organisms. At an initial 1% substrate concentration, the co-culture produced 200-587% more algal biomass than the axenic C. minutissima cultures. Co-cultures grown on 1% substrate consumed 23-737% more of the available carbon substrate than the sum of substrate consumed by E. coli and C. minutissima alone. At 1% substrate, total lipid and starch productivity were elevated in co-cultures compared to axenic cultures indicating that bacterial contamination was not detrimental to the production of biofuel precursors in this specific case. Bio-fouling of the reactors observed in co-cultures and acid formation in all mixotrophic cultures, however, could present challenges for scale-up.

Growing algae on anaerobic digestate could decrease the algal production cost while reducing nutrient pollution. In past work, we developed a successful biological pretreatment for digestate that enables rapid algal growth on digestate without dilution. The objective of this work was to test the effectiveness of this pretreatment in outdoor algae cultures over a year-long timeframe. The study was conducted in semi-continuous, replicated bubble column photobioreactors in Auburn, AL, USA. Algae could grow successfully in pretreated digestate during the fall, spring, and summer, achieving average growth rates of 30, 42, and 66 mg L-1 d-1, respectively. Although the cold temperatures in winter suppressed algal growth, external heating was not required to keep the cultures alive. For two weeks during the summer, the system was challenged with 100% digestate that did not undergo pretreatment, and the algal community suffered a culture collapse with a significant (p < 0.001) decrease in productivity compared to the previous batches in which pretreatment was used. Nitrogen, phosphorus, and sulfur removal were observed during robust algal growth. There was no nitrification in the photobioreactors during the initial 200 days, but evidence of nitrification emerged during the summer and persisted into the fall. Nitrospirae were detected by 16S rRNA sequencing, proving that nitrifying bacteria could adapt to high ammonium (462 - 1502 mg/L). The eukaryotic community was dominated by Coelastrum (>90%) in the cold season, and the dominance transitioned to Chlorella in the warm season (>95%) based on 18S rRNA sequencing. The low relative abundance of cyanobacteria showed that green algae were the favored photosynthetic organisms in the system. [ABSTRACT FROM AUTHOR]

Algal growth in water treatment plants can lead to the introduction of toxin and odour compounds from cyanobacteria and causes filter fouling. Here, we investigate the use of woven polypropylene covers and a proprietary copper sulfate formulation (EarthTec) to prevent algal growth within mesocosms designed to simulate settling basins. Chlorophyll a, soluble nutrients, weather patterns and cyanobacterial gene abundance were measured. The covers suppressed algal and cyanobacterial growth in mesocosms, likely due to a combination of light blockage and prevention of insects from entering the mesocosms. In contrast, mesocosms amended with EarthTec exhibited little difference from the control mesocosms in terms of algal growth. qPCR analysis revealed that only a small fraction of cyanobacteria in the system contained genes necessary for the production of the microcystin toxin. Despite differences between mesocosms and full‐scale settling basins, the data here suggest that woven covers can suppress algal growth while minimising evaporative water losses. [ABSTRACT FROM AUTHOR]

Wastewater treatment is an energy‐intensive process and a net emitter of greenhouse gas emissions. A large fraction of these emissions is due to intensive aeration of aerobic bacteria to facilitate break‐down of organic compounds. Algae can generate dissolved oxygen at levels in excess of saturation, and therefore hold the potential to partially displace or complement mechanical aeration in wastewater treatment processes. The objective of this study was to develop an internally consistent experimental and modeling approach to test the hypothesis that algal photosynthetic aeration can speed the removal of organic constituents by bacteria. This framework was developed using a simplified wastewater treatment process consisting of a model bacteria (Escherichia coli), a model algae (Auxenochlorella protothecoides), and a single carbon source that was consumable by bacteria only. This system was then tested both with and without the presence of algae. A MATLAB model that considered mass transfer and biological kinetics was used to estimate the production and consumption of O2 and CO2 by algae and bacteria. The results indicated that the presence of algae led to 18–66% faster removal of COD by bacteria, and that roughly one‐third of biochemical oxygen demand was offset by algal photosynthetic aeration. [ABSTRACT FROM AUTHOR]

Irradiation of aqueous solutions containing the sodium salt of sulfonated poly(ether etherketone) and HCO2H/HCO2− buffers with 350 nm photons induced photoreduction of CCl3F to CHCl2F and Cl−. In situ determinations of chloride ion concentrations enabled evaluation of the quantum yield, ϕ(Cl−), which amounted to 0.46 in air-free, neutral solutions. A delayed formation of F− was noticed with a quantum yield of 0.18 probably due to a secondary transformation. High concentrations of the halide ions inhibited somewhat the photoreduction, whereas phase-separated CFC droplets increased the reaction rate. Despite the high quantum efficiencies obtained, no post-irradiation formation of Cl− occurred. This observation together with other findings indicated that a chain process was not involved in the transformation. Most of the kinetic features of the photoreaction are explained in terms of a mechanism involving radicals of the reactants and of the polymer. Efficient photoreductions of CCl3F also took place in sulfonated poly(ether etherketone) solutions containing air. [ABSTRACT FROM AUTHOR]